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Research Article

Sildenafil Inhibits Chronically Hypoxic Upregulation of Canonical Transient Receptor Potential Expression in Rat Pulmonary Arterial Smooth Muscle

¹Johns Hopkins University ²Guangzhou Medical University

Submitted 10 December 2008 ; revised 26 October 2009 ; accepted in final form 30 October 2009

In pulmonary arterial smooth muscle cells (PASMCs), Ca²⁺ influx through store-operated Ca²⁺ channels thought to be composed of canonical transient receptor potential (TRPC) proteins is an important determinant of intracellular free calcium concentration ([Ca²⁺]_i) and pulmonary vascular tone. Sildenafil, a type V phosphodiesterase inhibitor that increases cellular cGMP, is recently identified as a promising agent for treatment of pulmonary hypertension. We previously demonstrated that chronic hypoxia (CH) elevated basal [Ca²⁺]_i in PASMCs due in large part to enhanced store-operated Ca²⁺ entry (SOCE); moreover, ex vivo exposure to prolonged hypoxia (4% O2 for 60 h) upregulated TRPC1 and TRPC6 expression in PASMCs. We examined the effect of sildenafil on basal [Ca²⁺]_i, SOCE, and the expression of TRPC in PASMCs under prolonged hypoxia exposure. We also examined the effect of sildenafil on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle (PA) from rats that developed chronically hypoxic pulmonary hypertension (CHPH). Compared to vehicle control, treatment with sildenafil (300 nM) inhibited prolonged hypoxia induced increases of: 1) basal [Ca²⁺]_i; 2) SOCE; 3) mRNA and protein expression of TRPC in PASMCs. Moreover, sildenafil (50mg/kg/day) inhibited mRNA and protein expression of TRPC1 and TRPC6 in PA from chronically hypoxic (10% O₂ for 21 days) rats, which were associated with decreased right ventricular pressure and right ventricular hypertrophy. Furthermore, we found, in PASMCs exposed to prolonged hypoxia, knockdown of TRPC1, or TRPC6 by their specific small interference RNA attenuated the hypoxic increases of SOCE and basal [Ca²⁺]_i, suggesting a cause and effect link between increases of TRPC1 and TRPC6 expression and the hypoxic increases of SOCE and basal [Ca²⁺]_i. These results suggest that sildenafil may alter basal [Ca²⁺]_i in PASMCs by decreasing SOCE through downregulation of TRPC1 and TRPC6 expression, thereby contributing to decreased vascular tone of pulmonary arteries during the development of CHPH.

Sildenafil; calcium signaling; TRPC; Pulmonary Arterial Smooth Muscle